Viral influenza, particularly the H5N1 strain of influenza A virus, is a serious threat to human health. Currently there are two different classes of approved anti-influenza drugs, which target different viral proteins: amantadine targets a protein called the M2 proton channel, while Tamiflu (oseltamivir) targets neuraminidase. In the last flu season, most strains of the influenza virus (including H5N1) have become resistant to the amantadine class of drugs, causing the CDC to issue a recommendation to discontinue their use. Thus, there is an urgent need for a second-generation amantadine-like drug that will be useful prophylactically against new mutant strains of the virus including H5N1. We will take a variety of approaches to design second generation M2 blockers. To accomplish this goal a team of investigators has been assembled (DeGrado, Cristian, Diamond, Klein, Lamb, Paessler, Pinto, Winkler), each with special expertise. This team, led by DeGrado, will build on important preliminary results to design second generation M2 blockers. To this end, we have crystallized the proton channel, both in the presence and absence of amantadine, and the structures will be solved in Aim 1. The availability of crystal structures of the protein will be used in structure-based drug design in Aim 2. In parallel, state-of-the-art high throughput screening methodologies will be employed to discover novel lead inhibitors (Aim 3). The in vitro potency of the lead compounds will be optimized in Aim 4. The goal of Aims 5 and 6 is to identify compounds or compound classes that demonstrate antiviral in vivo efficacy against M2 without obvious toxicity. These studies will evaluate acute toxicity, pharmacokinetic properties, metabolic stability and in vivo efficacy of the discovery lead compounds. The results from these studies will be used to select a few lead compounds for more complete drug development evaluations suitable for regulatory submission (Aims 7, 8). The ultimate goal of the program is to complete all studies to enable filing an IND. In addition to overseeing the overall program, DeGrado's group will be responsible for structural biology and the chemical aspects of the project.